Sathyanarayana N. Gummadi

Bio: Sathyanarayana N. Gummadi is an academic researcher from Indian Institute of Technology Madras. The author has contributed to research in topics: Phospholipid scramblase & Caffeine. The author has an hindex of 25, co-authored 139 publications receiving 2332 citations. Previous affiliations of Sathyanarayana N. Gummadi include Indian Institutes of Technology & University of Wisconsin-Madison.

Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine

Abstract: Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca2+) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni2+–NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca2+ ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques.

Biochemical evidence for lead and mercury induced transbilayer movement of phospholipids mediated by human phospholipid scramblase 1.

Abstract: Human phospholipid scramblase 1(hPLSCR1) is a transmembrane protein involved in bidirectional scrambling of plasma membrane phospholipids during cell activation, blood coagulation, and apoptosis in response to elevated intracellular Ca(2+) levels. Pb(2+) and Hg(2+) are known to cause procoagulant activation via phosphatidylserine exposure to the external surface in erythrocytes, resulting in blood coagulation. To explore its role in lead and mercury poisoning, hPLSCR1 was overexpressed in Escherichia coli BL21 (DE3) and purified using affinity chromatography. The biochemical assay showed rapid scrambling of phospholipids in the presence of Hg(2+) and Pb(2+). The binding constant (Ka) was calculated and found to be 250 nM(-1) and 170 nM(-1) for Hg(2+) and Pb(2+), respectively. The intrinsic tryptophan fluorescence and far ultraviolet circular dichroism studies revealed that Hg(2+) and Pb(2+) bind to hPLSCR1 and induce conformational changes. hPLSCR1 treated with protein modifying reagent N-ethylmaleimide before functional reconstitution showed 40% and 24% inhibition in the presence of Hg(2+) and Pb(2+), respectively. This is the first biochemical evidence to prove the above hypothesis that hPLSCR1 is activated in heavy metal poisoning, which leads to bidirectional transbilayer movement of phospholipids.

Bacterial extracellular polysaccharides involved in biofilm formation.

Abstract: Extracellular polymeric substances (EPS) produced by microorganisms are a complex mixture of biopolymers primarily consisting of polysaccharides, as well as proteins, nucleic acids, lipids and humic substances. EPS make up the intercellular space of microbial aggregates and form the structure and architecture of the biofilm matrix. The key functions of EPS comprise the mediation of the initial attachment of cells to different substrata and protection against environmental stress and dehydration. The aim of this review is to present a summary of the current status of the research into the role of EPS in bacterial attachment followed by biofilm formation. The latter has a profound impact on an array of biomedical, biotechnology and industrial fields including pharmaceutical and surgical applications, food engineering, bioremediation and biohydrometallurgy. The diverse structural variations of EPS produced by bacteria of different taxonomic lineages, together with examples of biotechnological applications, are discussed. Finally, a range of novel techniques that can be used in studies involving biofilm-specific polysaccharides is discussed.

The distribution and function of phosphatidylserine in cellular membranes.

Abstract: Phosphatidylserine (PS) is the most abundant negatively charged phospholipid in eukaryotic membranes. PS directs the binding of proteins that bear C2 or gamma-carboxyglutamic domains and contributes to the electrostatic association of polycationic ligands with cellular membranes. Rather than being evenly distributed, PS is found preferentially in the inner leaflet of the plasma membrane and in endocytic membranes. The loss of PS asymmetry is an early indicator of apoptosis and serves as a signal to initiate blood clotting. This review discusses the determinants and functional implications of the subcellular distribution and membrane topology of PS.